Sprays of charged liquid droplets can be produced through Electrospray and pneumatic nebulization or ultrasonic nebulization in the presence of an electric field. The mechanisms of ion production from unassisted Electrospray ionization have been described by Karbarle, P., J. Mass Spectrom. 35, 804-817 (2000)[1], and Karbarle, P. and Ho, Y. “Electrospray Ionization Mass Spectrometry”, Edited by Richard Cole, Chapter 1, 3-63, 1997[2]. The oxidation and reduction chemical reactions that occur, or that can be induced to occur, on conductive surfaces located in sample solution flow channels prior to or during the charged droplet formation process in Electrospray have been described by Van Berkel, G. L. “Electrospray Ionization Mass Spectrometry”, Edited by Richard Cole, Chapter 2, 65-105, 1997[3], Van Berkel, G. J., J. Am. Soc. Mass Spectrom. 2000, 11, 951-960[4] and Van Berkel, G. J., Asano, K. G. and Kertesz, V., Anal. Chem. 2002, 74, 5047-5056[5]. Promotion of oxidation or reduction of sample species on conductive surfaces during Electrospray ionization followed by mass spectrometric analysis can be a useful tool to enhance the sensitivity or aid in determining the structure of specific sample species. The production of ion species in sample solutions through reduction/oxidation reactions on surfaces in the first solution flow channel with solutions retaining a total net neutral charge prior to Electrospraying for mass spectrometrometric analysis has been reported by Hackett et. al., U.S. Pat. No. 5,869,832[6]. Commercially available products are available from ESA Inc., Chelmsford, Mass., that promote electrochemical reactions on surfaces in the sample solution flow path by applying voltages across electrodes extending into the sample solution flow. Specific electrode materials have been explored to control analyte oxidation in sample solutions prior to Electrospraying [5]. Split flow fractionation techniques used in conjunction with Electrospray ionization have been described by Van Berkel, U.S. Pat. No. 6,677,593 B1 [7] where electric or magnetic fields are applied across a sample solution in a flow path using two electrodes positioned on opposite sides of a sample solution flow path in contact with the solution flow to separate positive and negative ions into separate sample solution flow streams prior to charged droplet spraying. Van Berkel describes charged droplet spraying from such devices even without the presence of an external electric field applied at sample solution channel exit tips. Multiple Electrospray tips have been configured from a single sample solution flow channel by Kostianen and Bruins, Rapid Comm. in MS, Vol. 8, 549-558 (1994)[8]. Simultaneous Electrospraying of a sample solution from positive and negative sprays partitions the sample species in a manner that may not be readily predictable or controlled.
Neutral and charged species have been exchanged across membranes, transferred into and/or removed from sample solution flows to reduce or eliminate selected species in exchange for other selected species in a sample solution prior to Electrospraying. Acid and/or salt concentrations in a sample solution have been reduced by exchange across species specific semipermeable membranes prior to Electrospraying. Charged or neutral species are exchanged between a sample solution and a second solution through a semipermeable membrane driven by concentration gradients or electric fields maintained across such membranes while retaining an electrically neutral sample solution. In such devices electrodes are positioned in the first and second flow channels in contact with the sample solution and the second solution. Charged species in the sample and second solutions are neutralized through redox reactions occurring on the first and second flow channel electrode conductive surfaces resulting in a net neutral sample solution flow exiting these membrane devices. Such devices have been described and are sold by Dionex Corporation. The electric field is maintained across the membrane in these devices by applying a voltage difference between electrodes positioned in the first and second solution flow channels on either side of the membrane. The electric field applied across these electrodes drives the charged species across the membrane between the sample solution and second solutions. The electric field applied across the membrane in these devices is configured upstream and operated independent of a second electric field formed in the Electrospray process if these devices are interfaced to an Electrospray ion source through a connecting flow tube. The charged droplet spray current produced in these devices interfaced to an Electrospray probe is generated from redox reactions occurring at conductive surfaces located in the sample solution flow channel.
The present invention eliminates the occurrence of oxidation or reduction reactions on conductive surfaces in the sample solution flow channel during Electrospray Ionization (ES) while providing control of the total Electrospray current generated during the charged droplet formation process. The total Electrospray current has a direct impact on the size distribution of the charged droplets produced. In one embodiment of the invention, a sample solution flow channel is separated from a second solution or gas phase flow channel by a semipermeable membrane. The solution or gas composition flowing through the second flow channel can be varied as a step function or gradient during Electrospraying. In charged droplet sprayer embodiments configured according to the invention, the Electrospray field present at the sample solution spray tip during Electrospray is the only electric field driving charged species formation in the sample solution and second solution flow channels. Van Berkel, et. al. [5] describe the use of a cellulose ester 5000 Da molecular mass cutoff membrane membrane covering an electrode surface to prevent redox reactions of sample molecules on the electrode surface during the Electrospraying. The electrode is maintained at a kilovolt potential during Electrospraying, with an upstream grounded electrode positioned the sample solution flow path. No second solution is used behind the membrane in the reported Electrospray apparatus and no current measurement was taken on the grounded conductive surface in the sample flow path during Electrospray ionization to determine the extent of redox reactions occurring at the grounded electrode surface in the sample solution flow path. No explanation is given by the authors as to how the electrical contact is completed between the sample solution and the electrode through the membrane but it is likely that the sample solution wetting the membrane forms the electrical contact with the electrode maintained at kilovolt potentials during Electrospray ionization.
Severs, J. C., Harms, A. C., and Smith, R. D., Rapid Communications in Mass Spectrometry, Vol. 10, 1175-1178 (1996) and Severs, J. C. [9] and Smith, R. D., Anal. Chem. 1997, 69, 2154-2158 [10] describe a capillary electrophoresis (CE) Electrospray interface with a mass spectrometer (MS) in which a polysulfone dialysis membrane with a molecular weight cutoff of 10,000 Da separates the capillary electrophoresis solution from a second electrolyte solution in contact with a CE column exit electrode. In the CE/ES/MS interface described, the total Electrospray current is a small fraction of the total CE current flowing to the CE column exit electrode surface. In the CE runs reported, a +30 kV potential was maintained at the CE column entrance. In positive ion mode CE/ES operation reported, reduction occurs at the CE column exit electrode maintained at +1.6 kV as electrons pass from the electrode into the second electrolyte solution. During this CE/ES operation described, net positive charge transfers from the CE column solution into the second electrolyte solution through the membrane during positive Electrospray ionization. The net positive charge for charged droplet production in Electrospray appears to be supplied by a small portion of the electrophoretic charge moving from entrance to exit through the CE column driven by the 30 kV electrical potential applied at the CE column entrance. In the CE/ES apparatus described, the electric field maintained across the dialysis membrane is in the opposite direction required to supply charge for positive polarity Electrospray ionization. As described by Severs et. al. [9, 10] the second solution with electrolyte added is a static solution volume placed in a capillary tube surrounding the CE column exit end. The capillary tube has open ends to allow release of gas formed in redox reactions at the CE column exit electrode surface. The second electrolyte solution appears to remain in place due to surface tension of the liquid in the capillary tube. The authors report changing the second solution between CE/ES/MS runs, replacing the ammonium acetate solution with an acetic acid solution, resulting in a shift in charge state of multiply charged peaks appearing in mass spectrum of myoglobin and carbonic anhydrase. The shifting of the multiply charged profile to increased charge state peaks would occur with a reduction of pH in the CE solution. How this apparent decrease in pH occurs is not explained by the authors. The electric field applied across the membrane during CE/ES/MS with the apparatus described would have driven positively charged protons from the CE column solution into the second electrolyte solution effectively decreasing pH in the CE solution. One explanation could be that a portion of acetic acid in second solution remains in a neutral form and neutral acetic acid molecules may have transferred through the dialysis membrane into the CE solution driven by a concentration gradient during CE/ES/MS operation.
As described in the prior art, it may be desireable in some analytical applications to cause redox reactions with sample substances in solution prior to Electrospray MS analysis. However, for many applications it is preferable to minimize any changes to the analyte species in solution prior to Electrospraying to achieve minimum distortion of information regarding a solution composition in ES/MS analysis. In many applications including quantitative analysis, the study of peptides and proteins, high throughput screening, drug discovery, drug metabolite studies and biomarker detection it is preferred to have minimum modification of the analyte population during ES/MS analysis. The Electrospray probe apparatus configured according to the invention allows control of the Electrospray current using only the Electrospray electric field while preventing redox reactions from occurring on conductive surfaces in the first or sample solution flow path during Electrospray ionization. One embodiment of the invention provides control of the total Electrospray current and sample solution pH while preventing redox reactions from occurring on conductive surfaces in the sample solution flow path. This control of the Electrospray process allows optimization of ES/MS or ES/MSn analysis and expansion of ES/MSn or liquid chromatography Electrospray mass MS (LC/ES/MSn) analytical capability while insuring minimum modification of the analytes in the sample solution due to redox reactions prior to Electrospraying. The introduction of specific neutral or charged species into the sample solution through semipermeable membranes during Electrospray ionization can be selected and controlled to maximize ion signal for different classes of analyte compounds in the sample solution. The invention allows conducting of conductivity or pH scans during Electrospraying to maximize ion signal or to study processes occurring in solution such as protein folding as a function of pH. Preventing redox reactions from occurring on conductive surfaces in the first or sample solution flow path minimizes the carryover of contamination species that deplate from the conductive surfaces when the Electrospray polarity is changed. The contamination ions occurring in mass spectra when polarity is changed can reduce sample signal due to charge competition and cause interference peaks in the acquired mass spectrum. The charged droplet sprayer configured according to the invention reduces the time and solvent consumption required to flush sample solution flow paths, providing increased analytical throughput at lower cost per analysis.
The electrical circuit equivalence of conventional Electrospray ionization charged droplet formation and neutralization processes have been described by Kebarle, P., and Tang, L., Anal. Chem. 1993, 65, 972A-985A [11] and Jackson, G. S., and Enke, C. G., Anal. Chem. 1999, 71, 3777-3784 [12]. The total electrical current generated in unassisted or pneumatic nebulization assisted Electrospray is established by electrolytic processes occurring in solution. For a given voltage differential applied between the Electrospray tip and counter electrodes and for a given liquid flow rate, the total Electrospray current produced through the formation of charged liquid droplets is a strong function of the resistance, or inversely the conductivity, of the solution being Electrosprayed. The invention allows changing of the effective solution conductivity during Electrospraying by changing of the conductivity of a second solution flow separated from the sample solution flow by a semipermeable membrane. Charged species exchanged across the membrane between the first or sample solution and the second solution, effectively changing the conductivity of the sample solution, are driven across the membrane by the applied Electrospray electric field. Selected neutral species may also traverse the membrane driven by a concentration gradient between the first and second solutions that may also change the first solution conductivity. The controlled exchange of proton charged species across the membrane changes the first solution conductivity and pH. The invention allows the addition of protons or cations to the sample solution during positive polarity Electrospray ionization without the addition of the counter ion as is the case when acids or salts are added directly to the sample solution. The converse is true for negative polarity Electrospray ionization.
The total Electrospray current can be changed with precise and stable control during Electrospray ionization with no change to the charged droplet sprayer geometry or the applied Electrospray voltage. For a given solution flow rate, as the total Electrospray current increases, the size of the charged droplets produced decreases. Higher total Electrospray currents with smaller droplet size distributions allows faster drying of charged droplets and the reduction of aerosols produced from evaporating droplets with insufficient charge available to ionize non volatile components within the droplet. In unassisted Electrospray charged droplet production, each initial charged droplet breaks off with approximately half the Rayleigh limit of charge per droplet. For a given liquid flow rate, as the total ES current increases due to increasing solution conductivity, the total number of droplets produced must increase to carry the additional charge limited by the Rayleigh limit of charge per droplet. As the number of charged droplets produced per time increases, the charge to solution volume ratio increases. The same trends apply with pneumatic nebulization assisted Electrospray ionization charged droplet formation. Increasing the total charge available will increase analyte ES/MSn signal to the point where sufficient charge is available to ionize all analytic molecules. Increasing the total ES current beyond the equivalent analyte concentration may cause a decrease in ES/MSn signal. The charged droplet sprayer configured according to the invention allows rapid adjustment of total ES current during Electrospray ionization to maximize analyte signal in ES/MSn analysis.
Embodiments of the invention include charged droplet sprayers configured such that no redox reactions occur on conductive surfaces in the first or sample solution flow path during charged droplet formation in Electrospray ionization. In one embodiment of the invention, charged species are added to or removed from the first or sample solution through semipermeable, dielectric membranes separating the first solution from a second solution or gas flow. In this embodiment, the total charged droplet spray current produced from the charged droplet spraying process can be adjusted by modifying the second solution or gas phase composition, electric field strength across the membrane, electrode composition and geometry, membrane composition and geometry, the electric field at the spray tip, the number of spray tips, solution flow rate and other variables independent of the initial first or sample solution composition as will become apparent in the description of the invention. Through adjustment of such variables using the charged droplet sprayer configured according to the invention, charged droplet spraying can be optimized for a given application. For example, the amplitude of multiply charged peaks of proteins in a mass spectrum acquired by Electrospraying from an aqueous solution can be increased by adding protons through a fluorethylene polymer (Nafion™) dielectric membrane during Electrospraying using one embodiment of the invention. Alternative embodiments of the invention provide for charge separation and the addition or removal of net charge from the first or sample solution with all or a portion of the total charge droplet spray current generated through redox reactions occurring on conductive electrodes separate from the first solution flow path. Embodiments of the invention allow adjustment and optimization of charged droplet spraying for a given sample solution composition.